The need for catalysts that operate outside of their native context or which catalyze reactions that are not represented in nature has resulted in the development of "enzyme engineering" technology. The usual route taken in enzyme engineering has been a "rational design" approach, relying upon the understanding of natural enzymes to aid in the construction of new enzymes. Unfortunately, the state of proficiency in the areas of protein structure and chemistry is insufficient to make the generation of novel biological catalysts routine.
Recently, a different approach for developing novel catalysts has been applied. This method involves the construction of a heterogeneous pool of macromolecules and the application of an in vitro selection procedure to isolate molecules from the pool that catalyze the desired reaction. Selecting catalysts from a pool of macromolecules is not dependent on a comprehensive understanding of their structural and chemical properties. Accordingly, this process has been dubbed "irrational design" (Brenner and Lerner, PNAS USA 89: 5381-5383 (1992)).
Most efforts to date involving the rational design of enzymatic RNA molecules or ribozymes have not led to molecules with fundamentally new or improved catalytic function. However, the application of irrational design methods via a process we have described as "directed molecular evolution" or "in vitro evolution", which is patterned after Darwinian evolution of organisms in nature, has the potential to lead to the production of DNA molecules that have desirable functional characteristics.
This technique has been applied with varying degrees of success to RNA molecules in solution (see, e.g., Mills, et al., PNAS USA 58: 217 (1967); Green, et al., Nature 347: 406 (1990); Chowrira, et al., Nature 354: 320 (1991); Joyce, Gene 82: 83 (1989); Beaudry and Joyce, Science 257: 635-641 (1992); Robertson and Joyce, Nature 344: 467 (1990)), as well as to RNAs bound to a ligand that is attached to a solid support (Tuerk, et al., Science 249: 505 (1990); Ellington, et al., Nature 346: 818 (1990)). It has also been applied to peptides attached directly to a solid support (Lam, et al., Nature 354: 82 (1991)); and to peptide epitopes expressed within a viral coat protein (Scott, et al., Science 249: 386 (1990); Devlin, et al., Science 249: 249 (1990); Cwirla, et al., PNAS USA 87: 6378 (1990)).
It has been more than a decade since the discovery of catalytic RNA (Kruger, et al., Cell 31: 147-157 (1982); Guerrier-Takada, et al., Cell 35: 849-857 (1983)). The list of known naturally-occurring ribozymes continues to grow (see Cech, in The RNA World, Gesteland & Atkins (eds.), pp. 239-269, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1993); Pyle, Science 261: 709-714 (1993); Symons, Curr. Opin. Struct. Biol. 4: 322-330 (1994)) and, in recent years, has been augmented by synthetic ribozymes obtained through in vitro evolution. (See, e.g., Joyce, Curr. Opin. Struct. Biol. 4: 331-336 (1994); Breaker & Joyce, Trends Biotech. 12: 268-275 (1994); Chapman & Szostak, Curr. Opin. Struct. Biol. 4: 618-622 (1994).)
U.S. Pat. No. 5,807,718 (the disclosure of which is incorporated herein by reference) discloses a synthetic (i.e., non-naturally-occurring) catalytic DNA molecule (or enzymatic DNA molecule) capable of cleaving a substrate nucleic acid sequence at a defined cleavage site. That patent also discloses an enzymatic DNA molecule having an endonuclease activity. In various preferred embodiments, the catalytic DNA molecules of that patent are single-stranded. These catalytic DNA molecules can assume a variety of shapes consistent with their catalytic activity. Thus, in one variation, a catalytic DNA molecule includes one or more hairpin loop structures. In yet another variation, a catalytic DNA molecule may assume a shape similar to that of "hammerhead" ribozymes. An enzymatic DNA molecule of that patent can include a conserved core flanked by one or more substrate binding regions.
In another embodiment, the invention of that patent disclosed a non-naturally-occurring enzymatic DNA molecule comprising a nucleotide sequence defining a conserved core flanked by recognition domains, variable regions, and spacer regions.
Thus, in one preferred embodiment, the nucleotide sequence defined a first variable region contiguous or adjacent to the 5'-terminus of the molecule, a first recognition domain located 3'-terminal to the first variable region, a first spacer region located 3'-terminal to the first recognition domain, a first conserved region located 3'-terminal to the first spacer region, a second spacer region located 3'-terminal to the first conserved region, a second conserved region located 3'-terminal to the second spacer region, a second recognition domain located 3'-terminal to the second conserved region, and a second variable region located 3'-terminal to the second recognition domain.
The invention of that patent further disclosed methods of generating, selecting, and isolating enzymatic DNA molecules. In one variation, a method of selecting enzymatic DNA molecules that cleaved a nucleic acid sequence (e.g., RNA) at a specific site, included the following steps: (a) obtaining a population of synthetic, single-stranded DNA molecules; (b) admixing nucleotide-containing substrate sequences with the population of single-stranded DNA molecules to form an admixture; (c) maintaining the admixture for a sufficient period of time and under predetermined reaction conditions to allow single-stranded DNA molecules in the population to cause cleavage of the substrate sequences, thereby producing substrate cleavage products; (d) separating the population of single-stranded DNA molecules from the substrate sequences and substrate cleavage products; and (e) isolating single-stranded DNA molecules that cleave substrate nucleic acid sequences (e.g., RNA) at a specific site from the population.
Proteins and nucleic acids each have properties that offer unique advantages in performing catalytic transformations (Narlikar & Flerschlag, Ann. Rev. Biochem. 66: 19-59, 1997). Proteins possess a wide variety of functional groups that are suited to a broad range of chemical tasks, enabling protein enzymes to achieve extraordinary catalytic rate enhancements. Pancreatic ribonuclease A (RNase A), for example, catalyzes the cleavage of a dinucleotide substrate with a turnover rate of 1400 s.sup.-1 (delCardayr & Raines, Biochemistry 33: 6031-6037, 1994). Nucleic acids, although not endowed with the functional diversity of protein enzymes, are uniquely well suited for the sequence-specific recognition of nucleic acids through Watson-Crick base pairing. This capability allows nucleic acid enzymes to carry out chemical transformations on nucleic acid substrates with high sequence specificity and catalytic efficiency. In The RNA World. (Eds. R. F. Gesteland & J. F. Atkins), pp. 271-302. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. addition, the substrate-recognition domains of some nucleic acid enzymes can be altered without affecting catalytic activity, allowing them to operate in a general-purpose manner with nucleic acid substrates of almost any desired sequence.
Although nucleic acids are not endowed with diverse functional groups, their functional capabilities can be supplemented by the use of various metal and small-molecule cofactors. The activity of almost all known nucleic acid enzymes is dependent upon or greatly enhanced by divalent metal cations. In most cases, the metal is thought to participate directly in catalysis (for reviews, see Pan et al., 1993; Yarus, FASEB 7, 31-39, 1993; Steitz & Steitz, Proc Natl. Acad. Sci. USA 90: 6498-6502, 1993; Pyle, Science 261: 709-714, 1993; Joyce, Proc. Natl. Acad. Sci. USA 95: 5845-5847, 1998). In some cases, however, the metal appears to play an indirect role, perhaps increasing positive charge density within the active site or contributing to the structural integrity of the enzyme (Hampel & Cowan, Chem. Biol. 4: 513-517, 1997; Nesbitt et al., Chem. Biol. 4: 619-630, 1997; Young et al., Nucleic Acids Res. 25: 3760-3766, 1997; Suga et al., Biochemistry 37: 10118-10125, 1998; Murray et al., Chem. Biol. 5: 587-595, 1998).
The activity of one nucleic acid catalyst developed by in vitro selection is not dependent upon or even affected by the presence of divalent metal ions (Geyer & Sen, Chem. Biol. 4: 579-593, 1997). This metal-independent enzyme, however, exhibits a catalytic rate that is substantially lower than that of many of its metal-dependent counterparts. Another in vitro selected DNA enzyme operates without divalent metal in the presence of millimolar concentrations of histidine. The histidine cofactor is thought to serve as a general base in promoting the cleavage of an RNA phosphodiester (Roth & Breaker, Proc. Natl. Acad. Sci. USA 95: 6027-6031, 1997). The development of the latter catalyst suggests that nucleic acid enzymes could be made to utilize a wide variety of small-molecule cofactors, greatly expanding their functional capacity. On the other hand, the small molecule cofactor must be bound and correctly positioned for catalysis, which might require an enzyme of greater size and complexity.
The selection of nucleic acid catalysts that contain extended chemical functionality already built into the molecule has been made possible by the development of functionalized NTP analogues that are efficiently incorporated by polymerases. For example, replacement of UTP with a C5-imidazole-substituted UTP analogue enabled the in vitro selection of an imidazole-containing RNA enzyme that catalyzes amide bond formation (Wiegand et al., Chem. Biol. 4: 677-683, 1997). In another study, in vitro selection was used to develop a pyridine-functionalized RNA enzyme that catalyzes a Diels-Alder cycloaddition reaction (Tarasow et al., Nature 389: 54-57, 1997). Both the amide synthase and Diels-Alderase ribozymes require the functionally-enhanced nucleotides for their catalytic activity. In both cases, however, the role that the added functional groups play in catalysis has not yet been defined.
There are a variety of naturally-occurring RNA enzymes that have the ability to cleave RNA in a sequence-specific manner. These molecules have been used as "catalytic antisense" agents that can be directed to cleave target RNAs both in vitro and in vivo (for reviews, see Christoffersen & Marr, J. Med. Chem. 38: 2023-2037, 1995; Rossi, Biodrugs 9: 1-10, 1998). RNA enzymes obtained by in vitro evolution might also be used for this purpose (Vaish et al., Proc. Natl. Acad. Sci. USA 95: 2158-2162, 1998). Several years ago, the first example of a DNA enzyme was reported; a single-stranded DNA molecule obtained by in vitro selection that cleaves an RNA phosphodiester (Breaker & Joyce, Chem. Biol. 1: 223-229, 1994). More recently, a highly efficient, general-purpose RNA-cleaving DNA enzyme was developed (Santoro & Joyce, Proc. Natl. Acad. Sci. USA 94: 4262-4265, 1997). This molecule, composed of only .about.30 deoxynucleotides requires millimolar concentrations of a divalent metal cation for its catalytic activity. Compared to analogous RNA enzymes, the DNA enzyme is easier to prepare, is more resistant to chemical and enzymatic degradation, and exhibits more favorable kinetic properties (Santoro & Joyce, Biochemistry 37: 13330-13342, 1998). It has been applied to the cleavage of a variety of target RNAs, both in vitro (Unrau & Bartel, Nature 395: 260-263, 1998) and in vivo.
The lack of a 2' hydroxyl in DNA compared to RNA does not seem to be an impediment to efficient catalytic activity. It is intriguing to speculate, however, what catalytic functions DNA might be able to accomplish if it were endowed with some of the chemical groups that occur in proteins. The development of a family of dNTP derivatives that can be incorporated efficiently into DNA by polymerases (Sakthivel & Barbas, Aizgew. Chem. Int. Ed. 37: 2872-2875, 1998) has made it possible to address this question experimentally. As a test case, functionally enhanced DNAs were directed to cleave a target RNA substrate, withholding high concentrations of divalent metal cations, and instead providing imidazole-containing deoxyuridine residues and micromolar amounts of Zn.sup.2+. An imidazole moiety was chosen in order to confer the same chemical functionality that occurs in the amino acid histidine. Histidine residues are known to play a prominent role in the catalytic mechanism of many protein enzymes, especially ribonucleases and other phosphoesterases (for reviews, see Gerit, In Nucleases, 2nd Edition [Eds. S. M. Linn, R. S. Lloyd, & R. J. Roberts], pp. 1-34. Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1993; Lipscomb & Strater, Chem. Rev. 96: 2375-2433, 1996). The Zn.sup.2+ cofactor was chosen because of its propensity to coordinate to imidazole nitrogens, in both a structural and functional capacity. The resulting imidazole-containing DNA enzyme is a small, but highly efficient general-purpose endoribonuclease. This provides an experimental demonstration of a DNA enzyme that embodies the chemical functionality of a protein enzyme.